Combined Selective Plane Illumination Microscopy and FRAP maps intranuclear diffusion of NLS-GFP

摘要: Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics living cells. Its popularity is derived from widespread availability confocal microscopes relative ease experiment analysis. FRAP, however, limited ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) FRAP create SPIM-FRAP, wherein use a sheet light bleach 2D subsequently image same plane. This provides simultaneous quantification or every pixel given slice, thus moving measurements beyond these previous limitations. We demonstrate this technique by mapping intranuclear NLS-GFP live MDA-MB-231 cells; SPIM-FRAP proves be an order magnitude faster than correlation spectroscopy (FCS) based techniques such measurements. observe large length-scale (> ~500 nm) heterogeneity times NLS-GFP, which validated against simulated data sets. maps were correlated with images H2B address conflicting literature on role chromatin small molecules. observed no between histone density diffusion. developed simulation our experiments compare across techniques; measured coefficients are previously reported results, validating quantitative accuracy well-established methods. With recent rise accessibility SPIM systems, set provide simple quick means quantifying distribution